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Desarrollo de herramientas moleculares para la producción de policétidos y péptidos no ribosomales

AuthorsDel Cerro, Carlos
AdvisorGarcía, José Luis ; Galán, Beatriz
Metabolito secundario
Ingeniería genética
Issue Date2015
PublisherCSIC - Centro de Investigaciones Biológicas (CIB)
AbstractIntroduction: Marine environments are a huge source of natural compounds. Many of these molecules are synthesized as a defense mechanism by symbiotic microorganisms found in marine organisms such as sponges, tunicates and polychaetes. Among the wide diversity of natural products, the presence of molecules produced by bacterial biosynthetic clusters that encode polyketide synthases (PKS) and/or non-ribosomal peptide synthases (NRPS) is remarkable. The modular organization of these gene clusters, which sometimes cover large genome regions, allows the synthesis of compounds that can present a huge chemical variability. Depending on whether it concerns a culturable microorganism or a full microbiome, there are some differences in the tools used to identify PKSs and/or NPRSs gene clusters. If the producer microorganism has been isolated and cultured, some genomic tools such as massive sequencing procedures (including subsequent bioinformatics analysis) or genomic libraries cloned in high capacity vectors can be used. However, if the DNA sequence of interest belongs to a non-culturable microorganism, different metagenomics tools can be used. Among these techniques it is possible to find the generation of metagenomic libraries from environmental DNA and processes using massive sequencing that include analysis performed with specialized bioinformatics tools. Objectives: At the beginning of this PhD. Thesis we proposed the following objectives: 1. To develop genomic tools to optimize the production of molecules synthesized by genetic clusters enconding PKSs and/or NRPSs in culturable microorganisms. 2. To develop bioinformatics tools in order to identify sequences belonging to genetic clusters encoding PKSs or NRPSs in microbiomes. Results: Using genetic tools for the identification, characterization and heterologous expression of the didemnins biosynthetic gene cluster contained in Tistrella mobilis MES-10-09-028. To address the first objective of this Thesis, the culturable symbiont T. mobilis MES-10-09-028 was used as a proof of concept. First, massive sequencing procedures were used to obtain the T. mobilis MES-10-09-028 complete genome sequence. Further analysis allowed the didemnin biosynthetic gene cluster (ddn cluster) identification. The construction of the KR3 and DidA T. mobilis mutants, which are affected in the initial stages of the biosynthetic pathway of didemnins, showed that ddn cluster is responsible for the synthesis of didemnins, since both mutations block the didemnin production in MES-10-09-028 strain. Alternative intermediates from the synthesis were not detected in these mutants.
To achieve the heterologous production of didemnins, a BACs library using genomic DNA from MES-10-09-028 strain was constructed in Escherichia coli and a clone that contains all PKSs and NRPSs genes from ddn cluster was identified. In addition to this, the ddn cluster was efficiently expressed in the heterologous host. Using metagenomic tools for the identification of secondary metabolites biosynthetic clusters in non-culturable strains. To address the second objective of this Thesis, three different marine sponge samples were used, i.e., Polymastia littoralis, PMLT01 and Lithoplocamia lithistoides, in which antitumoral compounds were detected. Once microbiome DNA was isolated, environmental sequences were obtained using massive sequencing procedures. Taxonomic and functional information was obtained using bioinformatics tools with the metagenomic sequences. It was demonstrated that some symbionts are majority and therefore have to play an important metabolic role in sponges’ development. In silico tools were applied on metagenomic sequences for the detection of regions that belong to gene clusters enconding PKSs and/or NRPSs. As a result, biosynthetic clusters that can be related with antitumoral compounds production were detected. Conclusions: 1. T. mobilis MES-10-09-028 strain can be modified by genetic engineering tools, making it an excellent candidate for its use as a bacterial chassis for the production of PKSs and/or NRPSs related molecules. 2. It has been shown that ddn cluster is responsible for the synthesis of didemnins and it has been heterologously expressed in E. coli. 3. Analyzed marine sponges contain a few very major symbionts, which allows obtaining their complete genomic sequences using relatively low-depth massive sequencing procedures. 4. Analysis of gene sequences encoding PKS and/or NRPS clusters present in sponge metagenomes shows that antitumoral molecules detected are not always produced by the most abundant symbionts. 5. The analysis of G+C content of the contigs together with their respective abundance among the reads is a very powerful tool in order to easily assign sequences that belong to the same microorganism. In addition to this, the localization of the genetic cluster sequences that codify PKSs and/or NRPSs in this kind of data processing, allows us to predict the possible belonging of several sequences to the same cluster. 6. The aplidin synthesis could be originated by a subsequent modification of didemnin B once secreted. 7. The heterologous expression of NRPSs cluster depends on factors other than those related with the own genes of the cluster and, therefore, a better adaptation of the host is essential to achieve a successful production of these compounds. 8. The proportion of phage and mitochondrial sequences is specific to each sponge metagenome and may depend on factors related with intrinsic characteristics of each sample processing or other differences in the total abundance of microorganisms.
Description182 p.-64 fig.-18 tab.
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